WO2021150069A1 - Nouveau composé quinazoline utilisé comme agent thérapeutique contre des troubles métaboliques - Google Patents

Nouveau composé quinazoline utilisé comme agent thérapeutique contre des troubles métaboliques Download PDF

Info

Publication number
WO2021150069A1
WO2021150069A1 PCT/KR2021/000930 KR2021000930W WO2021150069A1 WO 2021150069 A1 WO2021150069 A1 WO 2021150069A1 KR 2021000930 W KR2021000930 W KR 2021000930W WO 2021150069 A1 WO2021150069 A1 WO 2021150069A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
present application
compound
acid
formula
Prior art date
Application number
PCT/KR2021/000930
Other languages
English (en)
Korean (ko)
Inventor
김종우
이치우
권혜신
권상훈
주욱일
유윤선
최보라
Original Assignee
주식회사 바이오웨이
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 주식회사 바이오웨이 filed Critical 주식회사 바이오웨이
Priority to EP21743962.9A priority Critical patent/EP4095140A1/fr
Priority to KR1020227029142A priority patent/KR20220159958A/ko
Priority to CN202180023754.7A priority patent/CN115380035A/zh
Priority to US17/794,651 priority patent/US20230097253A1/en
Publication of WO2021150069A1 publication Critical patent/WO2021150069A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/32—Nitrogen atom
    • C07D473/34—Nitrogen atom attached in position 6, e.g. adenine

Definitions

  • the present application relates to a novel quinazolinone compound that can be used for the treatment of metabolic diseases, particularly fatty liver.
  • Idelalisib (Formula 2) is a substance that has been developed as a blood cancer treatment and is used as a second-line drug for blood cancer.
  • Metabolic disorder is a disease that occurs in many modern people and poses a great risk to modern people's health. Various attempts have been made to treat metabolic diseases.
  • the inventors of the present application have arrived at the present application by confirming that the novel quinazolinone derivative exhibits therapeutic efficacy for metabolic diseases.
  • the quinazolinone derivative according to the present application has a structure similar to that of ideralisib, but exhibits very different properties from that of ideralisib and a different mechanism of action.
  • the present derivative molecule exhibits excellent efficacy in metabolic diseases, particularly lipid metabolic diseases, through different mechanisms of action.
  • This derivative molecule also showed excellent efficacy against Non-Alcoholic Steatohepatitis (NASH).
  • NASH Non-Alcoholic Steatohepatitis
  • a novel quinazolinone derivative is provided by the present application.
  • a pharmaceutical use of the quinazolinone derivative is provided by the present application. Furthermore, the use of the quinazolinone derivative for treating metabolic diseases is provided.
  • the present application provides a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof:
  • * is a stereocenter
  • the compound is an S-form or R-form based on the stereocenter, or a racemate in which the S-form and the R-form are mixed.
  • the present application provides a pharmaceutically acceptable salt of the compound represented by Formula 1, characterized in that the pharmaceutically acceptable salt is a hydrochloride salt.
  • a pharmaceutical composition for treating a lipid metabolic disease comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is provided:
  • * is a stereocenter
  • the compound is an S-form or R-form based on the stereocenter, or a racemate in which the S-form and the R-form are mixed.
  • the lipid metabolic disease is one selected from non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and liver fibrosis.
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • liver fibrosis liver fibrosis.
  • 1 is a protein expression level test result confirming the pharmacological mechanism of the compound according to the present invention.
  • FIG. 6 is an analysis of the results of FIG. 5 .
  • FIG. 8 is an analysis of the results of FIG. 7 .
  • FIG. 9 is a result confirming the expression of lipid production-related factors in steatosis-induced hepatocytes.
  • A shows the results of experiments on expression of factors related to lipid production according to the concentration of the compound of the present application.
  • (B) is the mRNA quantitative analysis result in Huh7 cells induced in steatosis.
  • 13 is a protein expression test result confirming the activity of the compound according to the present application as a multimodal modulator.
  • 16 is a PK confirmation result of the compound according to the present application.
  • 21 is a schematic diagram showing the pharmacological mechanism of the compound according to the present application.
  • FIG. 22 is an analysis of the results of FIG. 19 .
  • chemical structures are disclosed with corresponding chemical names. In case of a dispute, the meaning should be grasped by the chemical structure rather than the chemical name.
  • the term “pharmaceutically acceptable salt” refers to a salt that has the efficacy of a parent agent and is not biologically or undesirable (eg, non-toxic or not harmful to its receptor).
  • Suitable salts include, for example, a solution of the parent agent in an inorganic acid such as hydrochloric acid, phosphoric acid, sulfuric acid, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid.
  • acid salts which may be formed by mixing with a solution of a pharmaceutically acceptable acid such as an organic acid such as acid, glucuronic acid, trifluoroacetic acid or benzoic acid.
  • a pharmaceutically acceptable acid such as an organic acid such as acid, glucuronic acid, trifluoroacetic acid or benzoic acid.
  • pharmaceutically acceptable salts include alkali metal salts (such as sodium or potassium salts), alkaline earth metal salts (such as calcium or magnesium salts), and salts formed with suitable organic ligands such as quaternary ammonium salts.
  • the present application provides a therapeutic agent for a metabolic disease comprising a SIRT1 activator.
  • the present application provides a method of treating a metabolic disease, comprising prescribing a SIRT1 activator to a subject.
  • a molecule that functions as a SIRT1 activator is also provided by the present application in one embodiment.
  • the molecule according to the present application can function as an activator of SIRT1 as shown in the following experimental examples.
  • the activator of SIRT1 can function as a therapeutic agent for metabolic diseases as described below.
  • SIRT1 is a NAD-dependent deacetylase (NAD-dependent deacetylase) and gene regulatory proteins (genes such as ChREBP, SREBP-1, PPAR ⁇ , PGC-1 ⁇ , and NF- ⁇ B) as well as histones. -regulatory protein) is known to regulate deacetylation and activity.
  • SIRT1 is known to be involved in fatty acid oxidation and hepatic fat metabolism.
  • Steatosis an early stage of fatty liver disease, is characterized by the accumulation of excess triglycerides in the form of fat globules in the liver.
  • One cause is that triglyceride-rich chylomicrons and free fatty acids enter the liver through transmembrane proteins.
  • Another cause is that high levels of glucose and insulin promote the production of new lipids, and transcription factors such as the transcription factors ChREBP and SREBP1c are activated, which activate lipogenic enzymes such as FAS, ACC1, SCD1, and ELOVL6 in the liver.
  • fatty acid beta oxidation through the PPAR ⁇ /PGC-1 ⁇ signaling pathway in mitochondria, and the other is to convert triglycerides into blood in the form of very low-density lipoprotein (VLDL).
  • VLDL very low-density lipoprotein
  • SIRT1 When SIRT1 is activated, it can deacetylate ChREBP and SREBP1c to block the expression of lipogenesis-related genes, thereby inhibiting new lipogenesis (lipogenesis), and deacetylating PPAR ⁇ /PGC-1 ⁇ to increase fatty acid beta oxidation (Ding).
  • RB Bao J, Deng CX. Int J Biol Sci. (2017) 13(7):852-867).
  • liver cell-specific SIRT1 deletion impairs PPAR ⁇ signaling and reduces fatty acid beta-oxidation.
  • a high-fat diet was fed to a liver-specific SIRT1 knockout mouse, hepatic steatosis and hepatic inflammation were observed.
  • the SIRT1 activating substance can be used for the prevention or treatment of fatty liver and fatty liver-related diseases.
  • SIRT1 activating substances are known to be effective in preventing or improving symptoms of obesity, NAFLD, and diabetes.
  • resveratrol 3,4',5-trihydroxystilbene
  • Resveratrol is a polyphenol compound found in plants such as grapes and has a molecular formula of C 14 H 12 O 3 .
  • the effect of resveratrol on the prevention or treatment of heart disease, cancer, diabetes, and non-alcoholic fatty liver disease (NAFLD), etc. is being studied.
  • the present application provides a therapeutic agent for metabolic diseases comprising an AMPK activator.
  • the present application provides a method of treating a metabolic disease comprising prescribing an AMPK activator to a subject.
  • a molecule that functions as an AMPK activator is also provided by the present application in one embodiment.
  • the molecule according to the present application can function as an activator of AMPK, as shown in the following experimental examples.
  • the activator of AMPK can function as a therapeutic agent for metabolic diseases as described below.
  • AMPK AMP-activated protein kinase
  • AMPK functions to maintain intracellular energy homeostasis, and is known to promote glucose and fatty liver uptake and oxidation when intracellular energy is low.
  • AMPK is activated when energy in cells decreases due to metabolic stress or exercise, and inhibits the process of consuming ATP (fatty acid synthesis, cholesterol synthesis, etc.) and inhibits the process of ATP production (fatty acid oxidation, glycolysis, etc.)
  • AMPK is known to inhibit lipid production by participating in the phosphorylation of lipid production-related factors, Acetyl-CoA carboxylase (ACC) and Sterol regulatory element-binding protein 1c (SREBP1c).
  • ACC Acetyl-CoA carboxylase
  • SREBP1c Sterol regulatory element-binding protein 1c
  • AMPK is hexokinase II (hexokinase II, a factor related to beta oxidation of fatty acids) II), peroxisome proliferator-activated receptor alpha (hereinafter PPAR ⁇ ), peroxisome proliferator-activated receptor ⁇ , peroxisome proliferator-activated receptor ⁇ coactivator-1 (PPAR ⁇ coactivator) It is known to promote beta oxidation by activating -1; PGC-1), UCP-3, cytochrome C and TFAM. Focusing on the function of AMPK, there was an idea to treat metabolic diseases through AMPK activation (Winder WW, Hardie DG. AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes. Am J Physiol 1999;277(1):E1-E10.)
  • the present application provides a therapeutic agent for metabolic diseases comprising an inflammatory inhibitor.
  • the present application provides a method of treating a metabolic disease comprising prescribing an inflammatory inhibitor to the subject.
  • a molecule that functions as an anti-inflammatory agent is provided by the present application.
  • the inflammation inhibitor is an inflammatory factor, for example, I ⁇ B kinase ⁇ (I ⁇ B kinase ⁇ ; hereafter referred to as IKK ⁇ ), nuclear factor- ⁇ B (nuclear factor kappa-light-chain-enhancer of activated B cells; hereinafter NF- ⁇ B), interleukin 6, or 8 (interleukin; hereinafter IL6 or IL8), monocyte chemoattractant protein 1 (MCP1 hereinafter), or tumor necrosis factor ⁇ (Tumor necrosis factor ⁇ ; hereinafter TNF ⁇ ) can be inhibited.
  • IKK ⁇ I ⁇ B kinase ⁇
  • NF- ⁇ B nuclear factor- ⁇ B
  • interleukin 6, or 8 interleukin
  • MCP1 monocyte chemoattractant protein 1
  • TNF ⁇ tumor necrosis factor ⁇
  • the molecule according to the present application may function as an anti-inflammatory agent as shown in the following experimental examples. This is also due to the activation of AMPK and SIRT1. Inflammatory inhibitors may function as therapeutic agents for metabolic diseases as described below.
  • Inflammation is generally a beneficial phenomenon for returning tissues from an abnormal state to a normal state.
  • some patients with metabolic diseases develop a state of chronic inflammation in which the tissues are continuously inflamed as the tissues are not restored to a normal state.
  • Prolonged inflammatory conditions can be harmful to the human body.
  • Changes in the composition of immune cells in tissues due to inflammation are being studied to cause major symptoms of metabolic diseases including diabetes.
  • diabetes it is said that the insulin secretion gland may be destroyed due to the cytokine secretion of inflammatory immune cells.
  • Kupffer cells are activated to induce immune cells and activate immune cells in the liver cells.
  • Double hepatic stellate cells are known to promote the secretion and degradation of extracellular matrix proteins including fibrous collagen.
  • the sustained activation of Kupffer cells and HSC cells is known to be one of the main causes of liver fibrosis (Bataller R, Brenner DA. Liver fibrosis [published correction appears in J Clin Invest. 2005 Apr;115(4):1100]. J Clin Invest) 2005;115(2):209-218.)
  • anti-inflammatory drugs have the potential to improve the symptoms of fibrosis caused by metabolic diseases including liver fibrosis.
  • the term “multiple mode regulator” refers to a drug or compound having two or more modes of action expected to treat a target disease.
  • the molecule according to the present application can function as a multimodal modulator for treating metabolic diseases.
  • the mechanism of action of the multimodal modulator may be selected from an activator of SIRT1, an activator of AMPK, and inhibition of inflammation, as described above.
  • the present application provides a therapeutic agent for metabolic diseases comprising a multimodal modulator.
  • the present application provides a method of treating a metabolic disease comprising prescribing a multimodal modulator to a subject.
  • molecules that function as multimodal modulators are also provided by the present application in one embodiment.
  • the multimodal modulator may function as an activator of SIRT1 and an activator of AMPK. In one embodiment, the multimodal modulator may function as an activator of SIRT1 and an inhibitor of inflammation. In one embodiment, the multimodal modulator may function as an activator of AMPK and an inhibitor of inflammation. In one embodiment, the multimodal modulator may function as an activator of SIRT1, an activator of AMPK, and an inhibitor of inflammation.
  • the present application provides a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
  • * is a stereocenter
  • the compound is an S-form or R-form based on the stereocenter, or a racemate in which the S-form and the R-form are mixed.
  • the wavy bond structure bound to the stereocenter is to show that, depending on the isomer, it can be a bond entering the plane or a bond coming out of the plane.
  • the pharmaceutically acceptable salt may be an acid salt.
  • the acid salt is an inorganic acid such as hydrochloric acid, phosphoric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, glucuronic acid, tri Organic acids such as fluoroacetic acid or benzoic acid include all forms that can be used by those skilled in the art.
  • the pharmaceutically acceptable salt may be a hydrochloride salt.
  • the molecule according to the present application shares a structure similar to the previously developed drug Idelalisib.
  • the structure of ideralisip is shown in Chemical Formula 2 below.
  • Idelalisib has been studied as a treatment for blood cancer and as an inhibitor of phosphoinositide 3-kinase (PI3K).
  • the inventor of the present application found that the combination of halogen or R 1 having a size equivalent to that in Formula 1 and the 'isopropyl' group introduced to the stereocenter carbon shows a different aspect of pharmaceutical efficacy than the conventional ideralisib. confirmed that That is, while idelalisib is limited to the efficacy of hematologic cancer, the quinazolinone derivative of the present invention having the above-described characteristics showed more characteristic pharmaceutical efficacy in fatty liver-related diseases.
  • This table of contents describes the stereocenter of the compound.
  • the stereocenter exhibits different properties depending on the type of the substituent substituted therefor (hereinafter, the stereocenter substituent) and the type of the isomer.
  • the stereocenter substituent is an isopropyl (isopropyl) moiety.
  • the structure of the stereocenter substituent has a great influence on the properties of the compound.
  • Duvelisib is a quinazolinone derivative in which the stereocentric substituent is methyl. Despite these differences, duvelisib showed very poor selectivity for the isoform of PI3K than that of ideralisib.
  • the inventors of the present application confirmed that, when the stereogenic center substituent is an isopropyl residue, the function of regulating SIRT1, AMPK, and inflammatory factors is excellent, unlike previously known. Taking this into consideration, the inventors of the present application have confirmed that the novel molecule has an excellent effect on metabolic diseases.
  • the stereocenter may be of two isomeric types (S-type, R-type).
  • S-type isomeric types
  • R-type isomeric types
  • the type of isomer has a great influence on the properties of the present compound.
  • the type of isomer can affect the cytotoxicity of a compound and the aspect of its mechanism of action.
  • the stereogenic center may be S-type or R-type.
  • the stereogenic center may be S-shaped.
  • the stereogenic center may be R-shaped.
  • the compound may be a racemate in which S-form and R-form are mixed based on the stereogenic center.
  • the present application provides a compound represented by the following formula (3) or a pharmaceutically acceptable salt thereof:
  • the present application provides a compound represented by the following formula (4) or a pharmaceutically acceptable salt thereof:
  • the present application provides use of at least one compound selected from Formulas 1, 3, and 4 for treating metabolic diseases.
  • the present application provides a pharmaceutical composition comprising at least one compound selected from Formulas 1, 3, and 4 above.
  • the pharmaceutical composition may include a pharmaceutically acceptable excipient.
  • the compound according to the present application, or a pharmaceutically acceptable salt thereof may be administered in various formulations according to oral and/or parenteral methods during clinical administration.
  • the compound or a pharmaceutically acceptable salt thereof is used as a filler, a weight agent, a binder, a wetting agent, a diluent such as a disintegrant, a surfactant, or at least one of excipients. and can be formulated.
  • formulations for oral administration containing the compound according to the present application or a pharmaceutically acceptable salt thereof as an active ingredient include tablets, pills, hard/soft capsules, solutions, suspensions, emulsions, granules, elixirs, There may be troches and the like.
  • the formulation for oral administration includes a diluent (eg, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycic), and a lubricant (eg, silica, talc, stearic acid) and magnesium or calcium salts thereof and/or polyethylene glycol).
  • the tablet may include at least one of a binder such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine.
  • the tablet may contain at least one of a disintegrant or a boiling mixture and/or an absorbent, a coloring agent, a flavoring agent, and a sweetening agent, such as starch, agar, alginic acid or a sodium salt thereof, depending on the case.
  • a pharmaceutical composition comprising the compound according to the present application or a pharmaceutically acceptable salt thereof as an active ingredient may be used as a parenteral administration method.
  • the parenteral administration method may be any one of a method of injection by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.
  • the formulation for parenteral administration containing the compound according to the present application or a pharmaceutically acceptable salt thereof as an active ingredient may be prepared as a solution or suspension by mixing it with water together with a stabilizer or buffer, which is an ampoule or vial unit. can be prepared in dosage form.
  • the formulation for parenteral administration containing the compound according to the present application or a pharmaceutically acceptable salt thereof as an active ingredient is an adjuvant such as a preservative, a stabilizer, a wetting agent or emulsification accelerator, a salt and/or a buffer for regulating osmotic pressure, and others. It may contain therapeutically useful substances and may be formulated according to mixing, granulation or coating in a conventional manner.
  • the pharmaceutical composition according to the present application may be prepared as a formulation for sustained release of a compound or a pharmaceutically acceptable salt thereof through a polymer excipient or the like.
  • composition of the present application may include at least one active ingredient capable of exhibiting the same or similar function in addition to the compound according to the present application or a pharmaceutically acceptable salt thereof.
  • a preferred dosage of the pharmaceutical composition of the present application may be appropriately selected according to the patient's condition and weight, the severity of symptoms, drug form, administration route, and period. For optimal efficacy, it may be desirable for the pharmaceutical composition of the present application to administer the active ingredient in an amount of 0.2 mg/kg to 200 mg/kg per day. In addition, the pharmaceutical composition of the present application may be administered once a day or may be administered in several divided doses, but is not limited thereto.
  • the present application provides a method of treating a metabolic disease comprising administering to a subject one or more compounds selected from Formulas 1, 3, and 4 above.
  • the compound may be administered through an appropriate route, in the form of an appropriate composition, in an effective dose.
  • routes, compositions, and doses may be determined by means known in the art.
  • the present application provides a health functional food composition comprising at least one compound selected from Formulas 1, 3, and 4 above.
  • a compound of the present application or a pharmaceutically acceptable salt thereof, and a therapeutic use thereof are directed to a metabolic disease.
  • it targets those related to lipid metabolism among metabolic diseases.
  • the compound according to the present application can improve abnormal lipid metabolism because it has the effect of promoting lipid degradation and inhibiting lipid production.
  • provided by the present application is a pharmaceutical composition for treating a lipid metabolic disease.
  • the present application provides a nutraceutical composition for treating a lipid metabolism disease.
  • provided by the present application is a method of treating a metabolic disease associated with fat metabolism.
  • Exemplary lipid metabolic disorders include steatosis, liver disease, obesity, diabetes, hypertension, hypercholesterolemia, insulin resistance, and the like.
  • Exemplary liver diseases include fatty liver, such as alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD).
  • Nonalcoholic fatty liver includes simple fatty liver and non-alcoholic steatohepatitis (NASH), and complications due to fatty liver, e.g. liver fibrosis, cirrhosis, liver cancer, and esophageal varices.
  • NASH non-alcoholic steatohepatitis
  • Step 1 Preparation of tert -butyl (1- (5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl) -2-methylpropyl) carbamate
  • reaction mixture Cool to room temperature, extract with ethyl acetate and water.
  • organic layer is dehydrated with anhydrous magnesium sulfate (MgSO 4 ) and concentrated under reduced pressure.
  • the residue is purified by column chromatography, and the obtained solid is dried and tert -butyl (1- 5.72 g of (5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-2-methylpropyl)carbamate was obtained in 81% yield.
  • Step 3 Preparation of 2-(1-((7H-purin-6-yl)amino)-2-methylpropyl)-5-chloro-3-phenylquinazolin-4(3H)-one
  • Step 1 Preparation of (S)-tert-butyl(1-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-2-methylpropyl)carbamate
  • reaction mixture is cooled to room temperature and extracted with ethyl acetate and water.
  • organic layer is dehydrated with anhydrous magnesium sulfate (MgSO4) and concentrated under reduced pressure.
  • MgSO4 anhydrous magnesium sulfate
  • the residue is purified by column chromatography, and the obtained solid is dried (S 0.18 g of )-tert-butyl(1-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-2-methylpropyl)carbamate in 37% yield got it
  • Trifluoroacetic acid (3.75 mL) was mixed with (S)-tert-butyl(1-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-2-methylpropyl ) was added to a dichloromethane (15 mL) solution in which carbamate (0.54 g) was dissolved.
  • the reaction solution was stirred at room temperature for 0.5-1 hour, and then the pH of the reaction solution was adjusted to about 10 using aqueous ammonia.
  • the reaction mixture was extracted with dichloromethane and water, and the organic layer was dehydrated and filtered using anhydrous magnesium sulfate (MgSO4).
  • MgSO4 anhydrous magnesium sulfate
  • Step 3 Preparation of (S)-2-(1-((7H-purin-6-yl)amino)-2-methylpropyl)-5-chloro-3-phenylquinazolin-4(3H)-one
  • Step 1 Preparation of (R)-tert -butyl(1-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-2-methylpropyl)carbamate
  • reaction mixture is cooled to room temperature and extracted with ethyl acetate and water.
  • organic layer is dehydrated with anhydrous magnesium sulfate (MgSO 4 ) and concentrated under reduced pressure.
  • the residue is purified by column chromatography, and the obtained solid is dried (R) -tert -butyl(1-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-2-methylpropyl)carbamate in 74% yield 1.03 g was obtained.
  • Trifluoroacetic acid (7 mL) was mixed with (S) -tert -butyl (1- (5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-2-methylpropyl) Carbamate (1.03 g) was added to a dissolved dichloromethane (28 mL) solution.
  • the reaction solution was stirred at room temperature for 0.5-1 hour, and then the pH of the reaction solution was adjusted to about 10 using aqueous ammonia.
  • the reaction mixture was extracted with dichloromethane and water, and the organic layer was dehydrated and filtered using anhydrous magnesium sulfate (MgSO 4 ).
  • Step 3 Preparation of (R)-2-(1-((7H-purin-6-yl)amino)-2-methylpropyl)-5-chloro-3-phenylquinazolin-4(3H)-one
  • Huh7 cells were cultured in an incubator set at 37 ° C., humidity 95%, CO 2 5%.
  • Huh7 cells were added to the plate using growth medium (Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 ⁇ g/ml of streptomycin). After proliferation to 80-90%, it was used for the experiment.
  • growth medium Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 ⁇ g/ml of streptomycin.
  • Huh-7 cells in each well except for the control cells were treated with 2% BSA-bound PA at a concentration of 250 ⁇ M except for the control cells, and the control cells were pretreated with 2% FA-free BSA.
  • Each well was treated with 0.25 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M, and 40 ⁇ M of the R-, S-form of BW-3290, the compound of the present application, and then incubated for 24 hours.
  • Control cells and PA treated cells were treated with DMSO. All medium was aspirated and cells were gently washed 3 times with cold PBS.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • PVDF polyvinylidene fluoride
  • the transfer sandwich (sequence is paper, gel, membrane, and paper) was assembled and checked to ensure that no air bubbles were trapped in the transfer sandwich.
  • the transfer sandwich was placed in a transfer tank (Bio-Rad Laboratories) and an ice cube was placed in the transfer tank to prevent the transfer buffer from heating. Proteins were transferred from the gel to the PVDF membrane at 100 V for 30 min.
  • the membrane was incubated in a buffer in which the primary antibody was appropriately diluted.
  • the membranes of Fatty Acid Synthase (FAS), phosphor-AMP-activated protein kinase (p-AMPK), SIRT1, and SIRT3 were washed 3 times with TBST for 5 minutes each.
  • the membrane was incubated for 1 hour at room temperature with the recommended dilution of HRP (horseradish peroxidase)-conjugated secondary mouse antibody in blocking buffer.
  • HRP horseradish peroxidase
  • the membrane was washed 5 times with TBST for 5 min each. Excess reagent was removed and the membrane was covered with a clear plastic wrap.
  • ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) was applied according to the manufacturer's recommendations.
  • the compound of the present invention is a novel compound that can be used for the treatment of metabolic diseases and fatty liver according to the pharmacological mechanism of AMPK and SIRT1.
  • normal lung cells MRC, were cultured in an incubator set at 37°C and 95% humidity and 5% CO2.
  • CCK8 analysis was performed on the prepared MRC5 cells.
  • FIG. 2 is a view showing the experimental results confirming the cytotoxicity in MRC5 cells through CCK8 analysis.
  • the experimental results are presented only for the cells shown in FIG. 2, and the experimental results confirming the cytotoxicity in normal cells are not limited to the cells shown in FIG. 2, and cells corresponding to other normal cells are also normal. Cytotoxicity in cells can be confirmed. Experimental results will be described in more detail in the following description.
  • Huh7 cells were cultured in an incubator set at 37°C and 95% humidity and 5% CO2.
  • Huh7 cells were added to the plate using growth medium (Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 ⁇ g/ml of streptomycin). After proliferation to 80-90%, it was used for the experiment.
  • growth medium Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 ⁇ g/ml of streptomycin.
  • palmitic acid (PA) bound to 2% BSA was treated with 250 ⁇ M concentration in liver cells except for the control. After inducing steatosis, the fat cells were stained with Oil-Red-O staining.
  • Oil-Red-O coloring reagent (ORO solution is prepared by mixing 6 ml of ORO stock solution and 4 ml of distilled water, and then filtering through a 0.45 ⁇ m filter) to obtain fat globules staining. It proceeded for 30 minutes.
  • FIG. 3 shows that the inhibition of fat sphere formation of the compound according to the present application was confirmed through Oil-Red-O staining in comparison with the existing material.
  • the experimental results are presented only for the cells shown in FIG. 3, and the experimental results for confirming the effect of reducing fat accumulation in adipocytes are not limited to the cells shown in FIG. 3, but cells corresponding to cells derived from liver disease. Also, the effect of reducing fat accumulation in adipocytes can be confirmed.
  • the compound of the present application showed less fat cell staining compared to PA in all test groups.
  • the concentration of the compound 0.5 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, and 10 ⁇ M
  • the adipocyte staining was less than that of PA depending on the drug concentration.
  • the staining of fat globules is reduced not only at the same concentration of 10 ⁇ M but also at a lower concentration.
  • Huh-7 cells in each well except for the control cells were treated with 2% BSA-bound PA at a concentration of 250 ⁇ M except for the control cells, and the control cells were pretreated with 2% FA-free BSA.
  • the compound of the present application BW-3290
  • BW-3290 was treated with 0.5 ⁇ M, 2.5 ⁇ M, and 10 ⁇ M
  • the conventionally developed material PF-06409577 1 ⁇ M, 10 ⁇ M, elafibranor 10 ⁇ M, 30 ⁇ M was treated, and then cultured for 24 hours. did.
  • Control cells and PA treated cells were treated with DMSO. All medium was aspirated and cells were gently washed 3 times with cold PBS.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • PVDF polyvinylidene fluoride
  • the transfer sandwich (sequence is paper, gel, membrane, and paper) was assembled and checked to ensure that no air bubbles were trapped in the transfer sandwich.
  • the transfer sandwich was placed in a transfer tank (Bio-Rad Laboratories) and an ice cube was placed in the transfer tank to prevent the transfer buffer from heating. Proteins were transferred from the gel to the PVDF membrane at 100 V for 30 min.
  • the membrane was incubated in a buffer in which the primary antibody was appropriately diluted.
  • the membranes of P-ACC and LCAD were washed 3 times with TBST for 5 minutes each.
  • the membrane was incubated for 1 hour at room temperature with the recommended dilution of HRP (horseradish peroxidase)-conjugated secondary mouse antibody in blocking buffer.
  • HRP horseradish peroxidase
  • the membrane was washed 5 times with TBST for 5 min each. Excess reagent was removed and the membrane was covered with a clear plastic wrap.
  • ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) was applied according to the manufacturer's recommendations.
  • FIG. 4 is a diagram showing the results of drug screening compared to the existing substances under development for fat accumulation in liver cells induced steatosis. That is, it is a diagram showing the expression level of a protein related to fat synthesis inhibition and fatty acid oxidation. Since ACC is phosphorylated and p-ACC protein activity is lost, an increase in p-ACC means a decrease in fat synthesis. In addition, it can be confirmed that BW-3290, a compound of the present invention, has an equivalent or higher fat accumulation inhibitory effect in a concentration-dependent manner despite a lower concentration than PF-064099577 and Elafibranor.
  • the compound of the present invention has an improved fat accumulation inhibitory effect compared to PF-064099577 and Elafibranor.
  • 3TL-L1 mouse adipocyte progenitor cells (American Type Culture Collection) were cultured in an incubator set at 37° C., 95% humidity, and 5% CO 2 .
  • 3T3-L1 mouse adipocytes were sufficiently proliferated using a proliferation medium (Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine serum (BS), 100 U/ml of penicillin, and 100 ⁇ g/ml of streptomycin). Then, differentiation medium (DMEM containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 mM dexamethasone, and 5 ⁇ g/ml insulin) was treated for 2 days.
  • DMEM Dulbecco's modified Eagle's medium
  • BS bovine serum
  • streptomycin 100 ⁇ g/ml of streptomycin
  • induction medium (DMEM containing 10% FBS and 5 ⁇ g/ml insulin) was treated for 2 days for 2 days, and maintenance medium (DMEM containing 10% FBS) was applied daily to maintain differentiated adipocytes until the end of the experiment. replaced.
  • 3T3-L1 cells which are adipocytes, were differentiated into adipocytes and then fat cells were stained with Oil-Red-O was dyed.
  • Oil-Red-O coloring reagent is prepared by mixing 6 ml of ORO stock solution and 4 ml of distilled water and then filtration through a 0.45 ⁇ m filter) to stain lipocytes for 30 minutes. proceeded while
  • FIGS. 5 and 6 are diagrams showing the experimental results confirming the inhibition of lipocyte formation of the compound through Oil-Red-O staining.
  • the compound of the present application showed less adipocyte staining compared to differentiation in all test groups.
  • the concentration of the compound to 0.5 ⁇ M, 2.5 ⁇ M, 10 ⁇ M, and 20 ⁇ M, it can be confirmed that the adipocyte staining of the compound was less than that of differentiation in a drug concentration-dependent manner.
  • Huh7 cells were cultured in an incubator set at 37 ° C. humidity 95% and CO2 5%.
  • Huh7 cells were added to the plate using growth medium (Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 ⁇ g/ml of streptomycin). After proliferation to 80-90%, it was used for the experiment.
  • growth medium Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 ⁇ g/ml of streptomycin.
  • palmitic acid (PA) conjugated to 2% BSA was added to liver cells except for the control group 250. After inducing steatosis by treatment with ⁇ M concentration, fat cells were stained through Oil-Red-O staining.
  • hepatocytes were cultured to grow 80-90% in a 12-well plate, and the test substance was diluted in the culture medium induced to steatosis, except for the control group, and treated with 0.5 ⁇ M, 2.5 ⁇ M, and 10 ⁇ M.
  • Oil-Red-O coloring reagent (ORO solution is prepared by mixing 6 ml of ORO stock solution and 4 ml of distilled water, and then filtering through a 0.45 ⁇ m filter) to obtain fat globules staining. It proceeded for 30 minutes.
  • FIGS. 7 and 8 Experimental results confirming the effect of reducing fat accumulation in steatosis-induced Huh7 cells are shown in FIGS. 7 and 8 .
  • FIG. 7 is a view showing the experimental results confirming the inhibition of lipid sphere formation by BW-3290 through Oil-Red-O staining.
  • the experimental results are presented only for the cells shown in FIG. 7, and the experimental results for confirming the effect of reducing fat accumulation in adipocytes are not limited to the cells shown in FIG. 7, but cells corresponding to cells derived from liver disease. Also, the effect of reducing fat accumulation in adipocytes can be confirmed.
  • Huh-7 cells in each well except for the control cells were treated with 2% BSA-bound PA at a concentration of 250 ⁇ M except for the control cells, and the control cells were pretreated with 2% FA-free BSA.
  • Each well was treated with the compound of Formula 2 of the present application at 0.5 ⁇ M, 2.5 ⁇ M, and 10 ⁇ M, and then incubated for 24 hours.
  • Control cells and PA treated cells were treated with DMSO. All medium was aspirated and cells were gently washed 3 times with cold PBS.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • PVDF polyvinylidene fluoride
  • the transfer sandwich (sequence is paper, gel, membrane, and paper) was assembled and checked to ensure that no air bubbles were trapped in the transfer sandwich.
  • the transfer sandwich was placed in a transfer tank (Bio-Rad Laboratories) and an ice cube was placed in the transfer tank to prevent the transfer buffer from heating. Proteins were transferred from the gel to the PVDF membrane at 100 V for 30 min.
  • the membrane was incubated in a buffer in which the primary antibody was appropriately diluted.
  • the membranes of Sterol regulatory element-binding protein 1 (SREBP1) and Fatty Acid Synthase (FAS) were washed 3 times with TBST for 5 minutes each.
  • the membrane was incubated for 1 hour at room temperature with the recommended dilution of HRP (horseradish peroxidase)-conjugated secondary mouse antibody in blocking buffer.
  • HRP horseradish peroxidase
  • the membrane was washed 5 times with TBST for 5 min each. Excess reagent was removed and the membrane was covered with a clear plastic wrap.
  • ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) was applied according to the manufacturer's recommendations.
  • Huh-7 cells in each well except for the control cells were treated with 2% BSA-bound PA at a concentration of 250 ⁇ M except for the control cells, and the control cells were pretreated with 2% FA-free BSA.
  • Each well was treated with 0.5 ⁇ M of the compound of Formula 2 of the present application, and then incubated for 24 hours.
  • Control cells and PA treated cells were treated with DMSO. All medium was aspirated and cells were gently washed 3 times with cold PBS.
  • RNA lysis buffer was added.
  • the spin column was placed in a new Eppendorf tube and 50 ⁇ l of RNase-free water was added directly to the spin column membrane and centrifuged at 8,000 x g for 1 min to dissolve and isolate RNA.
  • RNA concentration was measured at 260 nm.
  • RNA reverse transcriptase 50 U
  • dNTP 4 mM dNTP 5 mM
  • magnesium chloride (MgCl 2 ) 5 mM RNase inhibitor 40 U 1 ⁇ g of total RNA was reverse transcribed in strand synthesis buffer.
  • cDNA was synthesized using a PCR (polymerase chain reaction) apparatus set to undergo denaturation at 95°C for 5 minutes, cDNA synthesis at 42°C for 50 minutes, and denaturation at 95°C for 5 minutes.
  • PCR polymerase chain reaction
  • the synthesized cDNA was diluted 1/10 and qRT-PCR was performed on the gene using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). (All primers for qRT-PCR were synthesized by GenoTech (Daejeon).)
  • a reaction mixture by mixing 3 ⁇ l of cDNA, 10 ⁇ l of Cyber Green (SYBR) master mix (Applied Biosystems), 3 ⁇ l of primer mix (sense primer and antisense primer), and 4 ⁇ l of distilled water (DW) for 15 seconds. Denaturation at 95° C., annealing at 60° C. for 1 min, and synthesis were subjected to 40 cycles.
  • SYBR Cyber Green
  • DW distilled water
  • the compound of the present invention reduces the expression of fat synthesis proteins and genes.
  • Huh-7 cells in each well except for the control cells were treated with 2% BSA-bound PA at a concentration of 250 ⁇ M except for the control cells, and the control cells were pretreated with 2% FA-free BSA.
  • Each well was treated with the compound of Formula 2 of the present application at 0.5 ⁇ M, 2.5 ⁇ M, and 10 ⁇ M, and then incubated for 24 hours.
  • Control cells and PA treated cells were treated with DMSO. All medium was aspirated and cells were gently washed 3 times with cold PBS.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • PVDF polyvinylidene fluoride
  • the transfer sandwich (sequence is paper, gel, membrane, and paper) was assembled and checked to ensure that no air bubbles were trapped in the transfer sandwich.
  • the transfer sandwich was placed in a transfer tank (Bio-Rad Laboratories) and an ice cube was placed in the transfer tank to prevent the transfer buffer from heating. Proteins were transferred from the gel to the PVDF membrane at 100 V for 30 min.
  • the membrane was incubated in a buffer in which the primary antibody was appropriately diluted.
  • the membranes of SIRT3, PGC1 ⁇ , PPAR ⁇ , and LCAD were washed 3 times with TBST for 5 minutes each.
  • the membrane was incubated for 1 hour at room temperature with the recommended dilution of HRP (horseradish peroxidase)-conjugated secondary mouse antibody in blocking buffer.
  • HRP horseradish peroxidase
  • the membrane was washed 5 times with TBST for 5 min each. Excess reagent was removed and the membrane was covered with a clear plastic wrap.
  • ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) was applied according to the manufacturer's recommendations.
  • the compound of the present invention As the concentration of BW-3290, the compound of the present invention, increased to 0.5 ⁇ M, 2.5 ⁇ M, and 10 ⁇ M, it can be seen that SIRT3, PPAR ⁇ , PGC1 ⁇ , and LCAD, which are proteins involved in fatty acid oxidation, increase in a concentration-dependent manner.
  • Huh-7 cells in each well except for the control cells were pretreated for 2 hours at a concentration of IL-1 ⁇ 10ng/ml except for the control to induce steatosis.
  • Each well was treated with the compound BW-3290 of the present application at 0.5 ⁇ M, 2.5 ⁇ M, and 10 ⁇ M, and then incubated for 24 hours. All medium was aspirated and cells were gently washed 3 times with cold PBS.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • PVDF polyvinylidene fluoride
  • the transfer sandwich (sequence is paper, gel, membrane, and paper) was assembled and checked to ensure that no air bubbles were trapped in the transfer sandwich.
  • the transfer sandwich was placed in a transfer tank (Bio-Rad Laboratories) and an ice cube was placed in the transfer tank to prevent the transfer buffer from heating. Proteins were transferred from the gel to the PVDF membrane at 100 V for 30 min.
  • the membrane was incubated in a buffer in which the primary antibody was appropriately diluted.
  • the membrane of P-NF ⁇ p-IKK ⁇ was washed 3 times with TBST for 5 min each.
  • the membrane was incubated for 1 hour at room temperature with the recommended dilution of HRP (horseradish peroxidase)-conjugated secondary mouse antibody in blocking buffer.
  • HRP horseradish peroxidase
  • the membrane was washed 5 times with TBST for 5 min each. Excess reagent was removed and the membrane was covered with a clear plastic wrap.
  • ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) was applied according to the manufacturer's recommendations.
  • RNA lysis buffer was added.
  • the spin column was placed in a new Eppendorf tube and 50 ⁇ l of RNase-free water was added directly to the spin column membrane and centrifuged at 8,000 x g for 1 min to dissolve and isolate RNA.
  • RNA concentration was measured at 260 nm.
  • RNA reverse transcriptase 50 U
  • dNTP 4 mM dNTP 5 mM
  • magnesium chloride (MgCl 2 ) 5 mM RNase inhibitor 40 U 1 ⁇ g of total RNA was reverse transcribed in strand synthesis buffer.
  • cDNA was synthesized using a PCR (polymerase chain reaction) apparatus set to undergo denaturation at 95°C for 5 minutes, cDNA synthesis at 42°C for 50 minutes, and denaturation at 95°C for 5 minutes.
  • PCR polymerase chain reaction
  • the synthesized cDNA was diluted 1/10 and qRT-PCR was performed on the gene using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). (All primers for qRT-PCR were synthesized by GenoTech (Daejeon).)
  • a reaction mixture by mixing 3 ⁇ l of cDNA, 10 ⁇ l of Cyber Green (SYBR) master mix (Applied Biosystems), 3 ⁇ l of primer mix (sense primer and antisense primer), and 4 ⁇ l of distilled water (DW) for 15 seconds. Denaturation at 95° C., annealing at 60° C. for 1 min, and synthesis were subjected to 40 cycles.
  • SYBR Cyber Green
  • DW distilled water
  • FIGS. 11 and 12 Experimental results confirming the expression levels of inflammatory factor proteins and genes in the inflammatory response-induced Huh7 cells are shown in FIGS. 11 and 12 .
  • 11 is a result of confirming the protein expression of P-NF ⁇ p-IKK ⁇ involved in the inflammatory response to Huh7 cells and the gene expression levels of IL-6, 8, and TNF- ⁇ .
  • the concentration of BW-3290 the compound of the present invention, increased to 0.5 ⁇ M, 2.5 ⁇ M, and 10 ⁇ M, it was confirmed that the expression of proteins and genes involved in the inflammatory response decreased in a concentration-dependent manner.
  • TNF ⁇ and IL-8 which are inflammation genes known as the cause of NASH, were identified, respectively, it was confirmed that the expression of the genes of TNF ⁇ and IL-8 was decreased, unlike the control group.
  • the compound of the present invention has a therapeutic effect even in fatty liver-related diseases such as NASH.
  • SIRT1 SIRT1, p53 conjugated AMC (aminomethylcoumarin) protein, and NAD+ 3 mM were added to diluted assay buffer (Tris-HCl 50 mM, pH 8.0, NaCl 137 mM, KCl 2.7 mM, and MgCl2 1 mM).
  • the reaction was started by adding 15 ⁇ l of a substrate solution containing 3 mM NAD+ to all wells used. Cover 96 wells with a plate cover and incubate on a shaker at room temperature (25 °C to 30 °C) for 45 min.
  • a stop/developing solution was prepared. To prepare a final 5ml solution, add 200 ⁇ l of nicotinamide (nicotinamide, NAM, Sigma-Aldrich) to 30 mg of the color powder and add 4.8 ml of diluted assay buffer until the color powder becomes a solution. stirred.
  • nicotinamide nicotinamide, NAM, Sigma-Aldrich
  • the plate cover was removed and 50 ⁇ l of the stop/chromic solution was added to each well.
  • the plate cover was removed and the plate read with Cytation 5 using an excitation wavelength of 360 nm and an emission wavelength of 450 nm.
  • the fluorescence of the background well was subtracted from the fluorescence of the 100% starting active well and sample well.
  • each sample value was subtracted from the 100% initial activity value, divided by the 100% initial activity value, and then the value multiplied by 100 was added to 100 and expressed as a percentage.
  • SIRT1 activation degree of sample (%) 100 + (initial activation value - sample value)/(initial activation value) ⁇ 100
  • Huh-7 cells in each well except for the control cells were treated with 2% BSA-bound PA at a concentration of 250 ⁇ M except for the control cells, and the control cells were pretreated with 2% FA-free BSA.
  • BSA-bound PA at a concentration of 250 ⁇ M except for the control cells
  • FA-free BSA 2% FA-free BSA
  • NaCl sodium chloride
  • EDTA ethylenediaminetetraacetic acid
  • pH 8.0 pH 8.0
  • HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
  • NP-40 non
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • PVDF polyvinylidene fluoride
  • the transfer sandwich (sequence is paper, gel, membrane, and paper) was assembled and checked to ensure that no air bubbles were trapped in the transfer sandwich.
  • the transfer sandwich was placed in a transfer tank (Bio-Rad Laboratories) and an ice cube was placed in the transfer tank to prevent the transfer buffer from heating. Proteins were transferred from the gel to the PVDF membrane at 100 V for 30 min.
  • the membrane was incubated in a buffer in which the primary antibody was appropriately diluted.
  • the membranes of P-AMPK and p-ACC were washed 3 times with TBST for 5 minutes each.
  • the membrane was incubated for 1 hour at room temperature with the recommended dilution of HRP (horseradish peroxidase)-conjugated secondary mouse antibody in blocking buffer.
  • HRP horseradish peroxidase
  • the membrane was washed 5 times with TBST for 5 min each. Excess reagent was removed and the membrane was covered with a clear plastic wrap.
  • ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) was applied according to the manufacturer's recommendations.
  • BW-3290 shows the results of confirming the SIRT1 activity of BW-3290. It can be seen that BW-3290 further increases the activity of SIRT1 when compared to a well-known control group (RSV, resveratrol) as a SIRT1 activator. This shows that BW-3290 directly binds to SIRT1 and increases its activity.
  • RSV control group
  • the concentration of the compound of the present invention As the concentration of the compound of the present invention, BW-3290, increased to 0.5 ⁇ M, 2.5 ⁇ M, and 10 ⁇ M, the concentration-dependently increased protein involved in SIRT1 and AMPK applied to multiple modes increases the activity of SIRT1, Even when the activity of AMPK is inhibited using an AMPK inhibitor, it can be confirmed that the activity of SIRT1 and AMPK is increased to act in a multimodal mode.
  • BW-3290 was formulated with a composition of 95% water containing 5% DMSO and 20% HPbCD, and was orally administered at a concentration of 10 mg/kg.
  • Blood samples were drawn from the jugular vein at time points of 0.25, 0.5, 1, 4, 8, 10, 12 and 24 hours.
  • the collected blood samples were centrifuged at 2500 x g at 4°C for 15 minutes to obtain plasma.
  • Bioanalysis for the measurement of pharmacokinetic parameters was analyzed by LC-MS/MS.
  • BW-3290 had excellent AUClast and Cmax, and it was confirmed that the concentration was maintained to some extent even after 12 hours after absorption.
  • the experimental example was performed in SanyalBio.
  • DIAMOND TM mouse model an animal model created using a Western Diet and Sugar Water, was used. DIAMOND TM mice were induced with WDSW for 14 weeks, 12 control mice and 12 BW-3290-treated mice were used, and BW-3290 was orally administered at 16 mg/kg once a day for 8 weeks. An autopsy was performed at 22 weeks, and blood collection and tissue storage were performed for each group individual.
  • body weight and liver weight were found to be decreased in the BW-3290 group compared with the control group (VC) ( FIG. 17 ). It was confirmed that the decrease in liver weight was slightly larger than body weight, which was confirmed to improve the Liver/body weight ratio.
  • ALT Alanine aminotransferase
  • AST Aspartate aminotransferase
  • ALP Alkaline phosphatase
  • ALT and AST which are mainly expressed in the liver, showed statistical significance and decreased compared to the control group.
  • BW-3290 a compound of the present invention, exhibits an effect of reducing body weight and liver weight, and it can be confirmed that it acts as a NASH therapeutic agent in an experiment using an animal model by showing a decrease in liver function enzymes.
  • the experimental example was performed in SanyalBio.
  • Hematoxylin & Eosin (H&E) staining and Oil-Red-O (ORO) staining were performed using liver tissue obtained after autopsy.
  • H&E staining is a basic staining method in histology, and compared to the control group, it was confirmed that the reduction of the fat cells that appear in the circle in the picture was shown in the BW-3290 treatment group, the compound of the present application (FIG.
  • the degree of staining of fat globules in liver tissue was confirmed through ORO staining, and it was confirmed that red-stained fat globules were decreased in the BW-3290 treatment group, the compound of the present application, compared to the control group (VC).
  • the experimental example was performed in SanyalBio.
  • liver tissue obtained after autopsy was stained with Sirius red and Masson's trichrome, which are staining methods that can confirm fibrosis.
  • liver fibrosis in the liver tissue is improved by the compound BW-3290 of the present application, and it can be confirmed through animal experiments that there is an effect of treating not only fatty liver and NASH, but also liver fibrosis.
  • the experimental example was performed in SanyalBio.
  • Insulin resistance test was performed one week before autopsy, and fasting was performed for 6 hours.
  • Insulin was injected intraperitoneally at a concentration of 0.75UU/kg, blood was drawn 15 minutes, 30 minutes, 45 minutes, and 90 minutes after injection, blood sugar was measured with OneTouchPlus Ultra Glucomer, and glucose score (glucose) score) was evaluated.
  • the compound of the present invention shows the possibility of BW-3290 acting not only as a NASH treatment agent but also as an insulin sensitizer based on the results of the insulin resistance test, and the possibility of expanding the indication to type 2 diabetes. show

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Les inventeurs sont parvenus à la présente demande en confirmant qu'un nouveau dérivé de quinazoline présente une efficacité thérapeutique contre des troubles métaboliques. Le dérivé de quinazoline selon la présente invention a une structure similaire à celle de l'idélalisib, mais présente des propriétés très différentes et un mécanisme d'action différent de celui de l'idélalisib. De plus, il a été montré que cette molécule dérivée présente une excellente efficacité contre des troubles métaboliques, en particulier des troubles du métabolisme des lipides, par l'intermédiaire du mécanisme d'action différent. Il a également été montré que la molécule dérivée présente une excellente efficacité contre la stéatohépatite non alcoolique (SHNA). Aucun médicament permettant de traiter la stéatohépatite non alcoolique n'avait été approuvé au moment du dépôt de la présente demande.
PCT/KR2021/000930 2020-01-23 2021-01-22 Nouveau composé quinazoline utilisé comme agent thérapeutique contre des troubles métaboliques WO2021150069A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP21743962.9A EP4095140A1 (fr) 2020-01-23 2021-01-22 Nouveau composé quinazoline utilisé comme agent thérapeutique contre des troubles métaboliques
KR1020227029142A KR20220159958A (ko) 2020-01-23 2021-01-22 대사질환 치료제로서의 신규한 퀴나졸리논 화합물
CN202180023754.7A CN115380035A (zh) 2020-01-23 2021-01-22 作为用于代谢紊乱的治疗剂的新型喹唑啉化合物
US17/794,651 US20230097253A1 (en) 2020-01-23 2021-01-22 Novel quinazoline compound as therapeutic agent for metabolic disorders

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020200009486A KR20210095495A (ko) 2020-01-23 2020-01-23 신규한 퀴나졸리논 화합물 및 이를 포함하는 약학적 조성물
KR10-2020-0009486 2020-01-23

Publications (1)

Publication Number Publication Date
WO2021150069A1 true WO2021150069A1 (fr) 2021-07-29

Family

ID=76992672

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2021/000930 WO2021150069A1 (fr) 2020-01-23 2021-01-22 Nouveau composé quinazoline utilisé comme agent thérapeutique contre des troubles métaboliques

Country Status (5)

Country Link
US (1) US20230097253A1 (fr)
EP (1) EP4095140A1 (fr)
KR (2) KR20210095495A (fr)
CN (1) CN115380035A (fr)
WO (1) WO2021150069A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060079538A1 (en) * 2004-05-13 2006-04-13 Dennis Hallahan Methods for inhibiting angiogenesis
WO2008064018A1 (fr) * 2006-11-13 2008-05-29 Eli Lilly & Co. Thiénopyrimidinones destinées au traitement de troubles inflammatoires et de cancers
WO2011011550A1 (fr) * 2009-07-21 2011-01-27 Calistoga Pharmaceuticals Inc. Traitement de troubles du foie par des inhibiteurs de pi3k
KR101932146B1 (ko) 2016-07-14 2018-12-24 주식회사 바이오웨이 Pi3k를 억제하는 신규한 퀴나졸리논 유도체 및 이를 포함하는 약학적 조성물
WO2019036430A1 (fr) * 2017-08-14 2019-02-21 Camp4 Therapeutics Corporation Procédés de traitement de maladies hépatiques
CN111840297A (zh) * 2020-08-24 2020-10-30 天津济坤医药科技有限公司 艾代拉里斯在制备用于治疗肝纤维化疾病的药物中的应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060079538A1 (en) * 2004-05-13 2006-04-13 Dennis Hallahan Methods for inhibiting angiogenesis
WO2008064018A1 (fr) * 2006-11-13 2008-05-29 Eli Lilly & Co. Thiénopyrimidinones destinées au traitement de troubles inflammatoires et de cancers
WO2011011550A1 (fr) * 2009-07-21 2011-01-27 Calistoga Pharmaceuticals Inc. Traitement de troubles du foie par des inhibiteurs de pi3k
KR101932146B1 (ko) 2016-07-14 2018-12-24 주식회사 바이오웨이 Pi3k를 억제하는 신규한 퀴나졸리논 유도체 및 이를 포함하는 약학적 조성물
WO2019036430A1 (fr) * 2017-08-14 2019-02-21 Camp4 Therapeutics Corporation Procédés de traitement de maladies hépatiques
CN111840297A (zh) * 2020-08-24 2020-10-30 天津济坤医药科技有限公司 艾代拉里斯在制备用于治疗肝纤维化疾病的药物中的应用

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
"The Cambridge Dictionary of Science and Technology", 1988
BATALLER RBRENNER DA: "Liver fibrosis [published correction appears", J CLIN INVEST, vol. 115, no. 4, April 2005 (2005-04-01), pages 1100
CHOI SEKWON SSEOK S ET AL., MOL CELL BIOL, vol. 37, no. 15, 2017, pages e00006 - 17
DING RBBAO JDENG CX, INT J BIOL SCI, vol. 13, no. 7, 2017, pages 852 - 867
HALEMARHAM, THE HARPER COLLINS DICTIONARY OF BIOLOGY, 1991
J CLIN INVEST, vol. 115, no. 2, 2005, pages 209 - 218
JEON SM: "Regulation and function of AMPK in physiology and diseases", EXP MOL MED, vol. 48, no. 7, 15 July 2016 (2016-07-15), pages e245
PURUSHOTHAM A ET AL., CELL METAB., vol. 9, no. 4, 2009, pages 327 - 38
SINGLETON ET AL.: "Dictionary of Microbiology and Molecular Biology", 1994
WINDER WWHARDIE DG: "AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes", AM J PHYSIOL., vol. 277, no. 1, 1999, pages E1 - E10, XP002364672
WING RRGOLDSTEIN MGACTON KJ ET AL.: "Behavioral science research in diabetes: lifestyle changes related to obesity, eating behavior, and physical activity", DIABETES CARE, vol. 24, no. 1, 2001, pages 117 - 123

Also Published As

Publication number Publication date
EP4095140A1 (fr) 2022-11-30
KR20220159958A (ko) 2022-12-05
KR20210095495A (ko) 2021-08-02
CN115380035A (zh) 2022-11-22
US20230097253A1 (en) 2023-03-30

Similar Documents

Publication Publication Date Title
AU2017374460B2 (en) Novel phenyl propionic acid derivatives and uses thereof
WO2012161518A2 (fr) Nouveaux dérivés de thio-urée utilisés en tant qu'activateurs de rorα, et composition pharmaceutique les contenant
WO2021162451A1 (fr) Composition pharmaceutique pour la prévention ou le traitement du cancer, contenant des acides biliaires ou des dérivés de ceux-ci, composés à base de biguanide, et deux ou plus de deux types d'agents antiviraux en tant que principes actifs
WO2021246797A1 (fr) Composition pharmaceutique pour la prévention ou le traitement d'un cancer et contenant un agent antiviral et un antidépresseur en tant que principes actifs
WO2020106119A1 (fr) Composition pharmaceutique comprenant des inhibiteurs de l'histone-désacétylase 6
WO2021182898A1 (fr) Composition anti-diabète et anti-obésité comprenant un nouveau composé
WO2021150069A1 (fr) Nouveau composé quinazoline utilisé comme agent thérapeutique contre des troubles métaboliques
WO2011122815A2 (fr) Nouveaux dérivés de quinoxaline
WO2015060613A1 (fr) Nouveau dérivé oxodihydropyridinecarbohydrazide antifongique
WO2012148140A2 (fr) Dérivés d'alcaloïdes à base d'imidazole ayant des effets d'inhibition de l'angiogenèse et antioxydants et leur procédé de préparation
WO2019190177A1 (fr) Dérivé de (2e,4e)-5-phényl-penta-2,4-dièn-1-one
WO2022075780A1 (fr) Composition pharmaceutique pour la prévention ou le traitement d'une vessie hyperactive
WO2017217741A1 (fr) Nouveau dérivé de furanone bromé, son procédé de préparation, et composition pharmaceutique comprenant le nouveau dérivé de furanone bromé en tant que principe actif
WO2021096314A1 (fr) Nouveau dérivé de benzimidazole et son utilisation
WO2017115914A1 (fr) Inhibiteur de la phosphorylation de ppary et composition pharmaceutique le comprenant
WO2021100897A1 (fr) Composition pharmaceutique pour la prévention ou le traitement du cancer, comprenant un composé à base de biguanide et du ferrocène ou un dérivé de ferrocène en tant que principes actifs
WO2021125905A1 (fr) Nouveau dérivé thiazole et son utilisation
WO2023085894A1 (fr) Nouveau dérivé de thio-urée utilisé en tant qu'activateur de rorα et composition pharmaceutique le comprenant
WO2022235097A1 (fr) Composition pharmaceutique contenant un nouveau dérivé de pyrazolo[3,4-b]pyridine pour la prévention ou le traitement d'une maladie métabolique comprenant l'obésité et le diabète sucré ou la stéatohépatite non alcoolique
WO2018194372A1 (fr) Nouvel activateur de sirt1 et utilisation médicinale correspondante
WO2023090908A1 (fr) Nouveau dérivé de flavonoïde et composition pharmaceutique le comprenant en tant que principe actif pour la prévention ou le traitement d'une maladie métabolique
WO2019235894A1 (fr) Composition pour la prévention ou le traitement du cancer solide comprenant un composé inhibant la liaison de aimp2-dx2 et de k-ras et nouveau composé inhibant la liaison de aimp2-dx2 et k-ras
WO2009145488A2 (fr) Protéine xbp1(s) agissant en tant que marqueur de différentiation d'adipocyte qui présente un moyen pour réguler une différentiation dans des adipocytes et application correspondante
WO2022225259A1 (fr) Composition anticancéreuse induisant la sénescence cellulaire et la mort cellulaire
WO2018016901A1 (fr) Composition pharmaceutique destinée à la prévention ou au traitement de maladies osseuses

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21743962

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021743962

Country of ref document: EP

Effective date: 20220823